A. Human Influenza Hemagglutinin Proteins
Influenza is a major, acute respiratory disease of human beings. It occurs in recurrent endemic and pandemic infections which start abruptly, spread rapidly and distribute frequently worldwide. Although the disease is usually relatively mild in healthy individuals, its results cause major financial losses from lost time at work and unaccountable impact in terms of pain and suffering. Thus, the prevention of outbreaks of influenza would be of great economic and social value.
The disease is caused by a virus vector which invades and infects host organism cells, disrupting their useful functions. Vaccines for use against influenza, prepared from killed virus, have been in use since the early 1940s. However, the usefulness of these vaccine products has been hampered by several problems such as:
(a) recipients of vaccine innoculations have not always reacted with protective effect,
(b) the potency of such vaccines has been variable from batch to batch and virus type to type,
(c) the administration of (frequently required) large amounts of vaccine produces adverse reactions often exceeding the tolerable limits of the human organism,
(d) their method of production from chick embryos (eggs) can cause incidental toxic effects because of unremovable egg impurities, and
(e) search for a suitable live attenuated influenza virus vaccine has not yet been successful because of possible reversion of such virus to wild type during administration into the human population.
These and other factors influence their widespread use and influenza remains as a dreadful disease and those individuals in the elderly age group and/or those who have chronic physical ailments are often susceptible to a greater degree. Thus, it would be very desirable to have a vaccine product for human influenza which, because of the method by which it is prepared, and its constituency, would overcome these problems.
The hemagglutinin (HA) protein is the most important protein involved in immunity against influenza virus. The hemagglutinin protein occurs as glycoprotein spikes on the virus surface. It has been shown to be structurally triangular and rod-shaped and is comprised of several subunits which contain the major antigenic and immunogenic determinants. The antigenic determinants are the antigenic binding sites for specific antibodies. The stimulated production of specific antibodies produces a state within the host organism of immunity to viruses containing the same antigenic determinants. Once induced, these antibodies can remain in the host organism for a significant period of time and later their production can be readily stimulated by reimmunization.
The various subunits of the hemaggluttinin protein are synthesized as part of a single polypeptide chain containing an amino acid precursor peptide attached at the N-terminus of the overall HA protein. Studies have shown that one of these units, referred to as HA1, which is located at the N-terminus of the uncleaved HA is the primary area of the HA molecule which contains the major antigenic determinants. There are a large number of strains and types of influenza viruses. They are distinct from one another by virtue of possessing variations in the antigenic determinants. These variations are referred to as "shifts" and "drifts" depending upon the extent of genetic variation. Thus, antibodies induced from one strain or type do not necessarily protect the host from a different strain. The variations in the antigenic determinants, referred to above, are due to mutations as well as reassortment in the viral genome that in turn lead to amino acid substitutions within the antigenic sites of the HA protein.
Reference is made to Structure and Variation in Influenza Virus, Proceedings of the International Workshop on Structure and Variation in Influenza Virus, Thredbo, Australia, Dec. 10-12, 1979, published by Elsevier North Holland, Inc., New York, N.Y., 1980, Editors: Graeme Laver and Gillian Air, in order to further illuminate the background of the present invention and to provide additional detail respecting its practice. By this reference, this citation is hereby incorporated herein.
Thus, it would be desirable to produce vaccines which can be readily modified to account for naturally occurring changes in the various viral strains, by focusing on the hemagglutinin protein itself, determining its, or at least its antigenic determinants', sequence(s) and preparing a vaccine containing the new or changed protein. The present mention provides the methods and means therefor.
B. Recombinant DNA Technology
With the advent of recombinant DNA technology, the controlled bacterial production of useful polypeptides has become possible. The workhorse of recombinant DNA technology is the plasmid, an extra-chromosomal loop of double-stranded DNA found in bacteria, oftentimes in multiple copies per bacterial cell. Included in the information encoded in the plasmid DNA is that required to reproduce the plasmid in daughter cells (i.e., a "replicon") and ordinarily, one or more selection characteristics, such as resistance to antibiotics, which permit clones of the host cell containing the plasmid of interest to be recognized and preferentially grown in selective media. The utility of plasmids, which can be recovered and isolated from the host microorganism, lies in the fact that they can be specifically cleaved by one or another restriction endonuclease or "restriction enzyme," each of which recognizes a different site on the plasmidic DNA. Thereafter heterologous genes or gene fragments may be inserted into the plasmid by endwise joining at the cleavage site or at reconstructed ends adjacent to the cleavage site.
As used herein, the term "heterologous38 refers to a gene not ordinarily found in, or a polypeptide sequence ordinarily not produced by, the host microorganism whereas the term "homologous" refers to a gene or polypeptide which is produced in the host microorganism, such as E. coli. DNA recombination is performed outside the microorganisms but the resulting "recombinant" plasmid can be introduced into microorganisms by a process known as transformation and large quantities of the heterologous gene-containing recombinant plasmid obtained by growing the transformant. Moreover, where the gene is properly inserted with reference to portions of the plasmid which govern the transcription and translation of the encoded DNA message, the resulting plasmid or "expression vehicle," when incorporated into the host microorganism, directs the production of the polypeptide sequence for which the inserted gene codes, a process referred to as expression.
Expression is initiated in a region known as the promoter which is recognized by and bound by RNA polymerase. In some cases, as in the trp operon discussed infra, promoter regions are overlapped by "operator" regions to form a combined promoter-operator. Operators are DNA sequences which are recognized by so-called repressor proteins which serve to regulate the frequency of transcription initiation at a particular promoter. The polymerase travels along the DNA, transcribing the information contained in the coding strand from its 5' to 3' end into messenger RNA which is in turn translated into a polypeptide having the amino acid sequence for which the DNA codes. Each amino acid is encoded by a unique nucleotide triplet or "codon" within what may for present purposes be referred to as the "structural gene," i.e. that part which encodes the amino acid sequence of the expressed product. After binding to the promoter, the RNA polymerase first transcribes nucleotides encoding a ribosome binding site, then a translation initiation or "start" signal (ordinarily ATG, which in the resulting messenger RNA becomes AUG), then the nucleotide codons within the structural gene itself. So-called stop codons, if present, are transcribed at the end of the structural gene whereafter the polymerase may form an additional sequence of messenger RNA which, because of the presence of the stop signal, will remain untranslated by the ribosomes. Ribosomes bind to the binding site provided on the messenger RNA, in bacteria ordinarily as the mRNA is being formed, and themselves produce the encoded polypeptide, beginning at the translation start signal and ending at the previously mentioned stop signal. The desired product is produced if the sequences encoding the ribosome binding site are positioned properly with respect to the AUG initiator codon and if all remaining codons follow the initiator codon in phase. The resulting product may be obtained by lysing the host cell and recovering the product by appropriate purification from other microorganism protein(s).
C. Promoter-Operator Systems
As examples, the beta lactamase and lactose promoter-operator systems have been commonly used to initiate and sustain the microbial production of heterologous polypeptides. Details relating to the make-up and construction of these promoter-operator systems have been published by Chang et al., Nature 275, 617-24 (1978) and Itakura et al., Science 198, 1056-63 (1977), which are hereby incorporated by reference. More recently, a system based upon tryptophan, the so-called trp promoter-operator system, has been developed. Details relating to the makeup and construction of this system have been published by Goeddel et al. Nucleic Acids Res. 8: 4057-74 (1980), which is hereby incorporated by reference.
The particular trp promoter-operator system has been constructed having a sequence of double-stranded DNA comprising a trp promoter-operator, nucleotides coding for the trp leader ribosome binding site, and in phase from a first 5' to a second 3' end of the coding strand, nucleotides encoding translation initiation for expression of a structural gene that encodes the amino acid sequence of the heterologous polypeptide. The DNA sequence referred to contains neither a trp attenuator region nor nucleotides coding for the trp E ribosome binding site. Instead, the trp leader ribosome binding site is efficiently used to effect expression of the information encoded by an inserted gene. Cells are transformed by addition of the trp promoter-operator-containing and attenuator-lacking plasmids and grown up in the presence of additive tryptophan. The use of tryptophan-rich media provides sufficient tryptophan to essentially completely repress the trp promoter-operator through trp/repressor interactions, so that cell growth can proceed uninhibited by premature expression of large quantities of heterologous polypeptide encoded by an insert otherwise under the control of the trp promoter-operator system. When the recombinant culture has been grown to the levels appropriate for industrial production of the polypeptide, on the other hand, the external source of tryptophan is removed, leaving the cell to rely only on the tryptophan that it can itself produce. The result is mild tryptophan limitation and, accordingly, the pathway is derepressed and highly efficient expression of the heterologous insert occurs, unhampered by attenuation because the attenuator region has been deleted from the system. In this manner, the cells are never severely deprived of tryptophan and all proteins, whether they contain tryptophan or not, can be produced in substantial yields. This system is described in more detail by Kleid et al., U.S. patent application Ser. No. 133,296, filed Mar. 24, 1980, which is hereby incorporated by reference.
D. Prior Art
Emtage et al., Nature (London) 283, 171 (1980), describe the gene sequence for the fowl plague virus and allude to its expression to produce the corresponding fowl plague hemagglutinin protein in a bacterial plasmid whose transcription is under control of a promoter derived from the tryptophan operon. See also German Offenlegungsschrift 3012303. In Structure and Variation in Influenza Virus, Supra., beginning on page 157, the same workers report on "The Cloning and Expression in E. coli of an Influenza Hemagglutinin Gene." In these publications, Emtage et al. essentially describe the construction of a plasmid vehicle that is designed for the expression of the hemagglutinin protein of fowl plague virus comprising sequences translated from dA:dT tails and linker, a prepeptide, the amino acid sequence of the entire fowl plague virus hemagglutinin comprised of some 560 amino acids and a tail of polypeptide derived from DNA of the pBR322 plasmid. The data presented suggest that the hemagglutinin molecule plus polypeptide presequence is expressed in some degraded form (or proteolytically cleaved form) by use of the tryptophan promoter-operator having an intact attenuator.